asms2004 label free quantitation

©2004 Waters Corporation

ASMS 2004

Towards Global Proteomics by Analysis of Exact Mass Retention Time Pairs: A proof-of-principle of the Quantitative Ion Mapping Technology

Scott J. Geromanos1; Keith Richardson1; Phillip Young1; Richard Denny1; Marc Gorenstein1; Guo-Zhong Li1; Timothy Riley1; Jeffrey Silva1; Gregory J. Opiteck2; Ashok R. Dongre2; and Stanley A. Hefta2 1Waters Corporation, Milford, MA; 2Bristol-Myers Squibb, Pennington, NJ

This poster illustrates the quantitative capabilities of the new Waters® Protein Expression System. The data presented were generated using commercially available tryptically digested human serum samples spiked with different con-centrations of five exogenous internal reference proteins contained in the Wa-ters Protein Expression System Standards. Data were acquired in alternate scanning low and elevated energy accurate mass LC/MS mode. Each spiked digest sample was analyzed in triplicate. The raw data was than processed through a suite of Protein Expression System Informatics tools to produce a list of unique Exact Mass Retention-Time (EMRT) components. The data are proc-essed further to match and compare the intensity of like EMRT components from different sample injections. Accurate mass low and elevated information associated with EMRT components that illustrate a significant change in inten-sity may optionally be exported to facilitate their qualitative identification.

Human serum (Sigma source) was tryptically digested according to the procedure described in Poster TPY 458 (C. Dorschel et al., “Protocols to Assure Reproducible Quantitative and Qualitative Analysis of Tryptic Digests of Complex Protein Mixtures for Global Proteomic Experi-ments”). Five aliquots of this human serum digest were spiked with Waters Protein Expression System Standards, containing equimolar levels of Yeast Enolase and Alcohol Dehydrogenase, Rabbit Glycogen Phosphorylate, Bovine Serum Albumin and Hemoglobin tryptic digest.

• LC/MS System: Waters Protein Expression System comprised of the Waters CapLC® System with the Waters Micromass® Q-Tof Ultima API Mass Spectrometer equipped with a NanoLockSpray™ Source operated at 12,000 mass resolving power

• Column: Waters NanoEase™ Atlantis™ dC18 Column, 300 µm x 15 cm • Mobile Phase: A = 1% Acetonitrile in Water, 0.1% Formic Acid, B = 80% Acetonitrile in

Water, 0.1% Formic Acid • Gradient: 6% to 40% B over 100 min. at 4.4 µL/min, followed by 10 min. rinse (99% B)

and 20 min. re-equilibration at initial conditions Each of the five spiked human serum (HS) digest samples was analyzed in triplicate. Injections were 5 microliters each and were made directly on-column. The total protein load for each in-jection was ~8.5 micrograms of Human Serum plus either 5.0, 1.0, 0.5, 0.25 or 0.1 pico-moles of the spiked Protein Expression System Standards. Exact mass LC/MS data was col-lected using an alternating low (10eV) and elevated (28eV to 35eV) collision energy mode of acquisition such that one cycle of low and elevated collision energy data was acquired every 4.0 seconds (Figure 1). The NanoLockSpray source was switched every 10 seconds to obtain a reference scan of [Glu1]-Fibrinopeptide lockmass calibrant. Figure 1. Data acquisition using the Waters Protein Expression System mass spectrometer

All results illustrated in the Figures and Tables are based on data collected in the low collision energy portion of the low and elevated energy data acquisition cy-cle. Figure 2 panels A through O illustrate the base peak intensity (BPI) chroma-tograms of the triplicate analyses of each of the five spiked HS digest samples. Note that the appearance of all 15 BPI chromatograms is quite similar indicating that the spiked protein digest components represent only a minor fraction of the total peptide content of each HS digest mixture. Figure 3 panels A through O illustrate the peak area integration of a non-lockmass corrected, selected ion chromatogram of an example spiked peptide (mass 978.49 eluting at approximately 77.5 minutes). Figure 4 panels A through O illustrate the complexity of a single lock mass cor-rected spectrum extracted at the apex of each selected ion chromatogram. Figure 5 panels A through O show an expanded mass range region of each of these spectra centered on m/z 978.49. The complexity of the spectra and the presence of significantly higher intensity ions indicate that the 978.49 EMRT is a minor component eluting at that point in the chromatogram. The software has ex-tracted the 978.49 EMRT component with excellent reproducibility and its linear-ity in response across the various spiked injection levels indicates that ion sup-pression effects are minimal. The mass precision for all 15 measurements of the 978.49 EMRT is +/- 4.64 ppm. Figures 6 through 9 compare data extracted from the fifteen spiked HS injec-tions using the Protein Expression System Informatics Software. Figure 6B illus-trates the log ratio of EMRT components from the 5.0 and 0.1 picomole spiked HS samples. The cloud of ions lying along the 45 degree axis represents EMRT peptides that were measured with an intensity ratio of approximately 1.0. These peptides are associated with HS peptides. The methodology employed allowed these ions to be measured over nearly 4 orders of magnitude in intensity. The cloud of ions lassoed in the upper left section of panel b illustrates the EMRT pep-tides associated with the spiked exogenous protein digests. This intensity ratio in-formation is further illustrated in Figure 6C as a frequency plot. The intensity of the two EMRT groupings clearly illustrate the 1:1 ratio of the HS peptides and the 1:50 ratio of the spiked HS peptides. Figure 6A illustrates that the coefficient of variation of the intensity measurements made on the HS peptides was generally less than 20%. Figures 7 through 9 compare 5.0 picomole vs. 0.5 picomole spiked samples, 0.50 versus 0.25 picomole spiked samples, and 0.25 versus 0.1 picomole spiked samples, respectively. A method for finding significant changes in peptide intensity is through the use of a paired Student t-Test. This test was employed to test the intensity of all the EMRT peptides that were observed to be in at least 2 out of 3 replicate injections in all of the spiked samples. This analysis produced a list of 63 EMRT peptides that had an intensity ratios between samples with a p-score lower than 0.01(data not shown). Figure 10 shows typical concentration plots for 6 randomly selected peptides. A quick perusal shows linearity across three orders of magnitude on peptides of varying ionization efficiencies. This list of peptides was searched against the SwissProt database using 10 ppm mass accuracy search criteria. Table 2 illustrates that 44 of the 63 peptides (>67%) mapped back to the five exogenous proteins spiked into the HS digest samples.

Figure 10. Concentration vs. Intensity Student t-Test Results

Human Serum + 5 picomolesWaters Protein Expression System

Standards

Figure 6.5 picomoles/100 femtomolesIn Human Serum

Figure 7. 5 picomoles/500 femtomolesIn Human Serum

Figure 8. 500 femtomoles/250 femtomolesIn Human Serum


Figure 2. BPI Chromatograms

A

B

C

Figure 3. SIC

978.xxxx

D

E

F

Human Serum + 1 picomoleStandards

G

H

I

Human Serum + 0.5 picomolesStandards

J

K

L


M

N

O


6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0

1 0 0

%

0 8 0 6 0 3 _ 4 9 7 H L _ Q u a n E x p S e r ia l_ 0 7 8 6 9 (7 7 .5 2 9 ) A M (C e n2 .2 2 e 47 9 0 .9 2 6 0

6 8 2 .7 0 1 0

6 6 9 .8 2 9 9

7 9 1 .4 2 4 7

8 2 0 .8 7 1 2

1 0 4 5 .9 8 9 41 0 4 6 .4 9 1 71 0 4 6 .9 9 4 8

6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0

1 0 0

%


6 8 2 .7 0 2 8

6 6 9 .8 3 0 0

7 9 1 .4 2 4 4

8 2 0 .8 7 1 9

1 0 4 5 .9 9 1 01 0 4 6 .4 9 2 31 0 4 6 .9 9 4 6

6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0

1 0 0

%


6 8 2 .7 0 0 1

7 9 1 .4 2 4 2

8 2 0 .8 7 2 1

8 2 1 .3 6 9 61 0 4 5 .9 8 9 78 2 1 .8 7 2 6

1 0 4 6 .4 9 1 11 0 4 6 .9 9 4 0

977 978 979 980 981m/z0

100

%

080603_497HL_QuanExpSerial_07 869 (77.529) AM (Cen3.72e3978.9915978.4916

977.5114

979.4922

979.9962980.4995

977 978 979 980 981m/z0

100

%


977.5165

979.4935

979.9962

980.5021

977 978 979 980 981m/z0

100

%

080603_497HL_QuanExpSerial_13 868 (77.397) AM (Cen4.02e3978.9918

978.4919

977.4807

979.4923

979.9935

980.4962

600 700 800 900 1000 1100m/z0

100

%

080603_497HL_QuanExpSerial_03 868 (77.388) AM (Cen,4, 85.00, Ar,12000.0,71.16e4820.8706

11559

682.70196038

682.36774011

683.03474309

821.370410147

821.87244900 937.4814

3465847.83232010

1023.55813184

1131.02591036

600 700 800 900 1000 1100m/z0

100

%


18903

682.369913883

669.83041592

683.034810713 820.8704

10078683.3688

4385937.9755

6199821.8717

4237

1023.55875929

1024.06013862

1131.0311605

600 700 800 900 1000 1100m/z0

100

%


13251682.7021

9377

682.36836462

628.24731074

683.03416139

821.369511558

821.87255308

937.97954841

847.83051770

1023.55753903

1131.0261885

977 978 979 980m/z0

100

%

080603_497HL_QuanExpSerial_03 869 (7727978.4924

977.5117977.9971

978.9933

979.4988

979.9883

977 978 979 980m/z0

100

%

080603_497HL_QuanExpSerial_09 869 (7689978.9965978.4922

977.5118978.0760

979.4936

979.9967

977 978 979 980m/z0

100

%


977.5134977.9865

979.4878

979.9998

600 700 800 900 1000 1100m/z0

100

%

080603_497HL_QuanExpSerial_04 870 (77.611) AM (Cen,4, 85.00, Ar,120002.92e4682.7063

682.3712

669.8302

683.0350

1023.5591820.8697683.3691

683.7068

937.9747821.8708 938.9804

1024.06181024.5626

600 700 800 900 1000 1100m/z0

100

%


682.3688

669.8305

820.8701683.0345

683.3688

683.7063

1023.5593937.9771

1024.06101024.5647

600 700 800 900 1000 1100m/z0

100

%


669.8304

683.0366

1023.5592937.9731683.3690

820.8691 938.9786

1024.0597

1024.56241131.0239

978 979 980 981m/z0

100

%

080603_497HL_QuanExpSerial_04 870 388978.4943

978.9926

979.4992

979.9919

980.4755

978 979 980 981m/z0

100

%


978.9942

979.4996

980.4976

978 979 980 981m/z0

100

%


978.9941

979.4996

979.9974

980.9815

978 979 980 981m/z0

100

%


132 979.520396

979.008472 980.4995

50

978 979 980 981m/z0

100

%


131 979.5164111

980.493047

978 979 980 981m/z0

100

%


108 979.503069

980.517844

980.038914

600 700 800 900 1000 1100m/z0

100

%

080603_497HL_QuanExpSerial_17 876 (78.097) AM (Cen,3, 80.001.70e4682.7003

16957

669.82902064

683.033110842 820.8694

8363683.36915064

937.97556609

1023.55806337

1131.0293;807

600 700 800 900 1000 1100m/z0

100

%


13092

669.82951592

683.03368515 820.8680

7303683.3688

38541023.5582

54241024.0614

3528

600 700 800 900 1000 1100m/z0

100

%


9871682.70106299

682.36884364

683.03274405

754.34521567

821.36958992

821.87184503

1023.55803239 1131.0281

1063978 979 980 981

m/z0

100

%

080603_497HL_QuanExpSerial_05 8173978.4968

173

979.5081111

980.4874;54

978 979 980 981m/z0

100

%


210

979.5123125

978 979 980 981m/z0

100

%


143

980.008449

Figure 4. Spectrum 550 –

1200 amu

Figure 5. Selected Precursor

A

B

C

A

B

C

A

B

C

A

B

C

Waters

®Pro

tein Ex

pressio

n Sy

stem In

form

atics

RMS error =4.64 ppm

CV Area =14.2%

A= Coefficient of Variation of Intensity lower [ ]

B= log10 Intensity higher [ ] vs. log10 Intensity lower [ ]

C= Intensity ratio histogram lower [ ]

vs. higher [ ]

Human Serum + 5 picomolesWaters Protein Expression System

Standards

Figure 6.5 picomoles/100 femtomolesIn Human Serum

Figure 7. 5 picomoles/500 femtomolesIn Human Serum



Figure 2. BPI Chromatograms

A

B

C

Figure 3. SIC

978.xxxx

D

E

F

Human Serum + 1 picomoleStandards

G

H

I


J

K

L


M

N

O


6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0

1 0 0

%


6 8 2 .7 0 1 0

6 6 9 .8 2 9 9

7 9 1 .4 2 4 7

8 2 0 .8 7 1 2

1 0 4 5 .9 8 9 41 0 4 6 .4 9 1 71 0 4 6 .9 9 4 8

6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0

1 0 0

%


6 8 2 .7 0 2 8

6 6 9 .8 3 0 0

7 9 1 .4 2 4 4

8 2 0 .8 7 1 9

1 0 4 5 .9 9 1 01 0 4 6 .4 9 2 31 0 4 6 .9 9 4 6

6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0

1 0 0

%


6 8 2 .7 0 0 1

7 9 1 .4 2 4 2

8 2 0 .8 7 2 1

8 2 1 .3 6 9 61 0 4 5 .9 8 9 78 2 1 .8 7 2 6

1 0 4 6 .4 9 1 11 0 4 6 .9 9 4 0

977 978 979 980 981m/z0

100

%


977.5114

979.4922

979.9962980.4995

977 978 979 980 981m/z0

100

%


977.5165

979.4935

979.9962

980.5021

977 978 979 980 981m/z0

100

%

080603_497HL_QuanExpSerial_13 868 (77.397) AM (Cen4.02e3978.9918

978.4919

977.4807

979.4923

979.9935

980.4962

600 700 800 900 1000 1100m/z0

100

%


11559

682.70196038

682.36774011

683.03474309

821.370410147

821.87244900 937.4814

3465847.83232010

1023.55813184

1131.02591036

600 700 800 900 1000 1100m/z0

100

%


18903

682.369913883

669.83041592

683.034810713 820.8704

10078683.3688

4385937.9755

6199821.8717

4237

1023.55875929

1024.06013862

1131.0311605

600 700 800 900 1000 1100m/z0

100

%


13251682.7021

9377

682.36836462

628.24731074

683.03416139

821.369511558

821.87255308

937.97954841

847.83051770

1023.55753903

1131.0261885

977 978 979 980m/z0

100

%

080603_497HL_QuanExpSerial_03 869 (7727978.4924

977.5117977.9971

978.9933

979.4988

979.9883

977 978 979 980m/z0

100

%


977.5118978.0760

979.4936

979.9967

977 978 979 980m/z0

100

%


977.5134977.9865

979.4878

979.9998

600 700 800 900 1000 1100m/z0

100

%


682.3712

669.8302

683.0350

1023.5591820.8697683.3691

683.7068

937.9747821.8708 938.9804

1024.06181024.5626

600 700 800 900 1000 1100m/z0

100

%


682.3688

669.8305

820.8701683.0345

683.3688

683.7063

1023.5593937.9771

1024.06101024.5647

600 700 800 900 1000 1100m/z0

100

%


669.8304

683.0366

1023.5592937.9731683.3690

820.8691 938.9786

1024.0597

1024.56241131.0239

978 979 980 981m/z0

100

%


978.9926

979.4992

979.9919

980.4755

978 979 980 981m/z0

100

%


978.9942

979.4996

980.4976

978 979 980 981m/z0

100

%


978.9941

979.4996

979.9974

980.9815

978 979 980 981m/z0

100

%


132 979.520396

979.008472 980.4995

50

978 979 980 981m/z0

100

%


131 979.5164111

980.493047

978 979 980 981m/z0

100

%


108 979.503069

980.517844

980.038914

600 700 800 900 1000 1100m/z0

100

%


16957

669.82902064

683.033110842 820.8694

8363683.36915064

937.97556609

1023.55806337

1131.0293;807

600 700 800 900 1000 1100m/z0

100

%


13092

669.82951592

683.03368515 820.8680

7303683.3688

38541023.5582

54241024.0614

3528

600 700 800 900 1000 1100m/z0

100

%


9871682.70106299

682.36884364

683.03274405

754.34521567

821.36958992

821.87184503

1023.55803239 1131.0281

1063978 979 980 981

m/z0

100

%


173

979.5081111

980.4874;54

978 979 980 981m/z0

100

%


210

979.5123125

978 979 980 981m/z0

100

%


143

980.008449

Figure 4. Spectrum 550 –

1200 amu

Figure 5. Selected Precursor

A

B

C

A

B

C

A

B

C

A

B

C

Waters

®Pro

tein Ex

pressio

n Sy

stem In

form

atics

RMS error =4.64 ppm

CV Area =14.2%

A= Coefficient of Variation of Intensity lower [ ]

B= log10 Intensity higher [ ] vs. log10 Intensity lower [ ]

C= Intensity ratio histogram lower [ ]

vs. higher [ ]

Introduction

Sample Preparation

Data Collection

Results and Discussion

• The protein digestion protocols employed in this study provide quantitatively reproducible results even on complex protein mix-tures as complex such as human serum.

• The analytical protocols employed in this study are capable of re-producibly measuring the intensity of peptides in complex protein digest mixtures over three to four orders of magnitude.

• Waters Protein Expression System Informatics is capable of ex-tracting peptide accurate mass, chromatographic retention time, and intensity information from complex protein digest mixtures in a quantitatively reproducible manner.

• The analytical protocols employed in this study demonstrate that the combination of exact mass and chromatographic retention time can provide a very unique signature for each peptide con-tained in a complex protein digest mixture.

• Waters Protein Expression System Informatics uses peptide exact mass and retention time signature information to match and quan-titatively compare the intensity of like identical peptides contained in protein digestion mixtures of a comparable control and experi-mental state.

Table 2. Peptide Mass Fingerprint Analysis of Student t-Test Results

1. 16/63 matches (25%). RABIT Name: Glycogen phosphorylase, muscle form (Myophosphor Index: 463045 MW: 97290 Da pI: 6.8 Sequence 979.5407 979.5464 -5.8 835 843 1 (R)LPAPDEKIP (-) 1117.5616 1117.5642 -2.3 415 425 0 (R)VAAAFPGDVDR (L) 1177.5519 1177.5530 -0.90 471 479 0 (K)DFYELEPHK (F) 1262.5911 1262.5945 -2.7 774 783 0 (K)VFADYEEYVK (C) 1278.5392 1278.5391 0.074 726 735 0 (R)GYNAQEYYDR (I) 1386.7427 1386.7494 -4.8 415 427 1 (R)VAAAFPGDVDRLR (R) 1440.7295 1440.7375 -5.5 522 533 0 (K)LLSYVDDEAFIR (D) 1442.6935 1442.6956 -1.5 279 290 0 (R)VLYPNDNFFEGK (E) 1550.7635 1550.7702 -4.3 508 520 0 (R)IGEEYISDLDQLR (K) 1566.7963 1566.7916 3.0 257 270 0 (K)DFNVGGYIQAVLDR (N) 1580.8292 1580.8324 -2.0 741 754 0 (R)QIIEQLSSGFFSPK (Q) 1663.8410 1663.8300 6.6 2Met-ox 603 618 1 (R)TVMIGGKAAPGYHM 1840.9268 1840.9234 1.9 279 293 1 (R)VLYPNDNFFEGKELR (L) 1853.9958 1853.9948 0.56 492 507 0 (R)WLVLCNPGLAEIIAER (I) 1874.9087 1874.9071 0.87 372 387 0 (K)TCAYTNHTVLPEALER (W) 1889.9995 1890.0085 -4.8 623 640 0 (K)LITAIGDVVNHDPVVGDR (L 2. 10/63 matches (15%). gi|30794280|ref|NP_851335.1| albumin [Bos taurus] Index: 351651 MW: 69324 Da pI: 5.8 Sequence 1163.6200 1163.6312 -9.6 66 75 0 (K)LVNELTEFAK (T) 1291.5995 1291.6026 -2.4 300 309 0 (K)ECCDKPLLEK (S) 1479.7871 1479.7960 -6.0 421 433 0 (K)LGEYGFQNALIVR (Y) 1502.6156 1502.6143 0.85 375 386 0 (K)EYEATLEECCAK (D) 1554.6537 1554.6535 0.14 387 399 0 (K)DDPHACYSTVFDK (L) 1576.7693 1576.7681 0.75 139 151 0 (K)LKPDPNTLCDEFK (A)

3. 6/63 matches (9%). YEAST Name: Enolase 1 (2-phosphoglycerate dehydratase) (2-ph Index: 213440 MW: 46802 Da pI: 6.2 Sequence 807.4390 807.4365 3.1 179 185 0 (K)TFAEALR (I) 1159.6121 1159.6111 0.82 186 195 0 (R)IGSEVYHNLK (S) 1373.6436 1373.6411 1.8 244 255 0 (K)IGLDCASSEFFK (D) 1416.7140 1416.7222 -5.8 16 28 0 (R)GNPTVEVELTTEK (G) 1578.7960 1578.8015 -3.5 89 103 0 (K)AVDDFLISLDGTANK (S) 1840.9268 1840.9227 2.2 33 50 0 (R)SIVPSGASTGVHEALEMR (D) 4. 6/63 matches (9%). YEAST Name: Alcohol dehydrogenase I (YADH-1) Acc. #: 171025 Species: UNREADABLE Name: gi|171025|gb|AAA344 Index: 343993 MW: 36823 Da pI: 6.3 Sequence 1071.6357 1071.6414 -5.3 225 234 1 (K)EKDIVGAVLK (A) 1136.5694 1136.5740 -4.1 9 18 0 (K)GVIFYESHGK (L) 1355.6079 1355.6088 -0.65 277 287 0 (K)CCSDVFNQVVK (S) 1386.7427 1386.7415 0.86 262 276 0 (R)ANGTTVLVGMPAGAK (C) 1447.7940 1447.8048 -7.5 320 332 0 (K)VVGLSTLPEIYEK (M) 2312.1337 2312.1482 -6.3 235 258 0 (K)ATDGGAHGVINVSVSEAAIEA 35. 6/63 matches (11%). gi|27819608|ref|NP_776342.1| hemoglobin, beta [beta globin] Acc. #: 122572 Species: BOVIN Name: Hemoglobin beta chain Index: 521112 MW: 15955 Da pI: 7.0 Sequence 1328.7186 1328.7174 0.90 17 29 1 (K)VKVDEVGGEALGR (L) 1422.7242 1422.7269 -1.9 120 131 0 (K)EFTPVLQADFQK (V) 1448.6836 1448.6844 -0.54 82 94 0 (K)GTFAALSELHCDK (L) 2089.9682 2089.9541 6.8 40 58 0 (R)FFESFGDLSTADAVMNNPK (V) 1177.6812 1177.6822 1.21 3 15 0 (K)VVAGVANALAHR(S) 821.4074 821.4094 2.27 1 7 0 (-) MLTAEEK(T)

Summary

asms2004 label free quantitation

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